Stuttering has long been known to have a genetic component. We have recently identified mutations in the GNPTAB gene that occur in individuals who stutter but do not occur in normal controls. Based on these results, we went on to identify mutations in two genes that function in the same biochemical pathway, GNPTG and NAGPA. Together these results indicate that stuttering can be associated deficits in the lysosomal targeting pathway. A goal of our current research is to identify how the subtle metabolic defects caused by mutations in these three genes lead to stuttering without any other discernible symptoms. In initial studies designed to localize the site of the pathology involved, we are measuring the level of expression of these three genes in more than 60 different regions of the human brain. We are also working to develop a mouse model of human stuttering. This is being done by creating so-called knock-in strains of mice that carry the mutations identified in humans who stutter. These experiments require a detailed acoustical analysis of mouse vocalization, which is largely ultrasonic in nature. In these experiments, we are working with Drs. Terra Barnes and Tim Holy at Washington University in St. Louis, who are leaders in the study of mouse vocalization. In a parallel line of inquiry, we are working with Dr. Stuart Kornfeld, also at Washington University in St. Louis. Dr. Kornfeld is a world leader in the study of the enzymes encoded by the GNPTAB, GNPTG, and NAGPA genes. The goal of these studies is to better understand the effects of the mutations observed in human stutterers on the function of these enzymes at the molecular and cellular level. In the past year we have performed additional family ascertainment and recruitment in Pakistan. This has resulted in the enrollment of six new consanguineous families, all characterized by a high density of individuals with persistent stuttering. Linkage analysis in these families has been completed, and we have discovered that a variant gene located on chromosome 3 is the cause of stuttering in one of these families. We are currently working to identify the causative gene at this locus. We have also continued our studies in a group of families in Cameroon, West Africa, in which stuttering occurs consistent with a simple inherited trait. We had previously obtained evidence that a gene on chromosome 1 is responsible in the largest of these families, but follow-up studies, including a subsequent new genome-wide linkage analysis failed to confirm this linkage. It is now clear that more than one causative gene (at more than one location) is at work in this large family, and we have identified significant linkage on chromosomes 15 and 3. We are working to refine these linkage locations and to identify candidate genes in these regions. Our studies of deficits in the sense of taste currently focus on sweet taste. During the past year, we have evaluated variation in other genes that encode known components of the mammalian taste transduction system. Variation in most of the candidate genes we've evaluated shows no evidence of association with variation in sweet taste perception in our subject population. However, we've obtained strong evidence that genetic variation in the G-alpha gustducin gene exerts a major effect on sweet perception, and further experiments to confirm this association, and to understand thee mechanism by which this variation exerts its effect, are underway. We are also taking a broader approach to identify novel components of the sweet taste perception machinery using a genome wide association study (GWAS) approach. Genotyping 600,000 single nucleotide polymorphism (SNP) variants distributed across the genome has identified a novel locus that shows association with sweet taste perception in a group of 165 normal human subjects. We are currently working to understand the mechanism by which variation in this gene affects sweet taste perception.